Browsing by Subject "CRISPR/Cas9"

Now showing 1 - 5 of 5
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    Bottom-up Pediatric Sarcoma Modeling Using Genetic Engineering and Induced Pluripotent Stem Cell Technologies
    (2022-12) Becklin, Kelsie
    From the onset, induced pluripotent stem cell (iPSC) technology radically changed how we study human disease and it continues to improve our understanding of disease ontology. Continual advances in cell culture techniques, genome engineering, and the -omics fields have expanded the use of iPSCs into our daily disease modeling toolkit. Using iPSCs to model cancer is not especially new; in fact, the first iPSC-derived from cancer cells was around 2011. But, the dedifferentiation process is largely inhibited in solid tumor cells and these cancer-derived iPSCs only capture a single cancer cell genome, losing all heterogeneity. Sarcomas are rare, heterogenous tumors arising from the mesenchymal lineage. Many sarcomas, such as Osteosarcoma (OSA) and Ewing sarcoma (ES), have had limited therapeutic advancements since the advent of chemotherapy. To improve therapeutic outcomes for these patients it is becoming clear that we need to identify tumor-promoting molecular profiles and gain a better understanding of tumor evolution. To do this, we implemented the use of genetic engineering strategies in karyotypically normal iPSC to generate bottom-up models of OSA and ES. OSA is the most common pediatric cancer of the bone and is characterized by a complex genome, but few bona fide OSA-dependent mutations have been identified. To make our iPSC-derived iOSA model, we installed OSA-associated mutations in TP53 and RB1 using the CRISPR/Cas9 system to generate knockout iPSC. After differentiation into mesenchymal stromal cells (iMSC) and osteoblasts (iOB) we then used retrovirus to overexpress constitutively active cMYCT58A and/or hRASG12V. The mutated iMSC and iOB cells had differential proliferation rates, colony forming ability, and tumor formation potential in immunodeficient mice, as well as evidence of large karyotype level mutations similar to those seen in human OSA genomics. Additionally, tumors from the iOSA model had RNA-seq profiles resembling primary OSA. This model demonstrates that using iPSCs for cancer modeling in genomically complex cancers is possible and can illuminate how these cancers initiate and evolve. In addition to OSA, I used iPSCs to model ES, a translocation driven cancer, with a 9-fold higher incidence rate in children of European (EUR) ancestry compared to African (AFR). Using iPSC-derived from individuals spanning the polymorphic spectrum of ES diagnosis, I initiated expression of the ES driving alteration, a EWSR1-ETS translocation. Specifically, I used lentivirus to express the EWSR1-FLI1 fusion protein (EWS/FLI) in iPSC-derived neural crest cells (iNCC). Cells of increasing AFR ancestry had lower tolerance to EWS/FLI expression, a result in line with the aforementioned differences in incidence of ES seen in EUR and AFR children. To investigate the molecular basis of this observation, we used RNA-seq and CUT&TAG to determine the gene expression and global occupancy differentials across ancestries driven by EWS/FLI. Genetic loci that were both differentially expressed and bound were nominated as our ancestry-linked differential Ewing sarcoma response (ALDER) loci. To this end we have identified 80 ALDER loci containing established and novel EWS/FLI target genes for further analysis. This study demonstrates the feasibility and utility of ancestry-informed iPSC modeling to identify novel and potentially targetable pathways to treat ES. In this work we applied genetic engineering tools in iPSC to generate novel models of the gnomically complex OSA, and the gnomically quiet, translocation driven ES. Collectively, the models described here provide a baseline system to study how OSA and ES initiate and the early stages of cancer development.
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    Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-mediated Counter-selection
    (2019-06) Domenech Corts, Anna
    Shewanella are invaluable hosts for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals, to create microbial fuel cells and for understanding extracellular electron transfer. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. In this dissertation, I describe development of simple and efficient genome editing tools for precise and site-directed mutagenesis of Shewanella. First, In Chapter I, I review recent advances in synthetic biology that accelerate the study and engineering of bacterial phenotypes. In chapter II, I show the development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of up to ~108 transformants/µg DNA and which is adaptable to other strains. Using this method for DNA transfer, in chapter III, I characterize a new phage recombinase, W3 Beta from Shewanella sp. W3-18-1 and show its utility for in vivo genome engineering (recombineering) using linear single-stranded DNA oligonucleotides. In my experiments the W3 Beta recombinase gives an efficiency of ~5% recombinants among total viable cells. In addition, I show the functionality of this new system in S. amazonensis, a strain with few genetic studies but of interest given its higher temperature range for growth and wide range of carbon sources utilized. In chapter IV, I demonstrate use of the CRISPR/Cas9 system as a counter-selection to isolate recombinants. When coupled to recombineering, this counter-selection results in an extremely high efficiency of >90% among total surviving cells, regardless of the gene or strain modified. This efficiency allows isolation of several different types of mutations made with recombineering, and even allows identification of rare recombinants that form independently of W3 Beta expression. This is the first effective and simple strategy for recombination with markerless mutations in Shewanella. With synthesized single-stranded DNA as substrates for homologous recombination and CRISPR/Cas9 as a counter-selection, this new system provides a rapid, scalable, versatile and scarless tool that will accelerate progress in Shewanella genomic engineering. Finally, I conclude in Chapter V with an overview of the challenges and future directions of the technologies demonstrated here, discussing possible advancements that could further enhance the study of Shewanella.
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    Genome engineering in large animals for agricultural and biomedical applications
    (2013-08) Tan, Wenfang
    Precision genetics will enhance genome-based improvement of livestock for agriculture and biomedicine. This thesis aimed to modify large animal genomes with precision; as the technologies progressed, our capability expanded from random insertional transgenesis to nucleotide-level precision. It began with Sleeping Beauty (SB) transposon mediated rapid integration of dominant negative Myostatin alleles. All piglets generated from treated cells harbored the transgenes; however, we were unable to study phenotypes due to death of the founder animals. We then sought to introgress a SNP into porcine Myostatin through recombinant Adeno-associated Virus (rAAV) mediated gene targeting. We achieved a 2x10-4 targeting frequency but only one-half of the targeted colonies harbored the SNP. Similarly, we succeeded in porcine LDLR gene knockout; however, targeted clones were often confounded by "bystander" cells with only random insertions of the targeting vector. We turned to develop TALENs for efficient targeting of important genes. TALENs demonstrated high activity in both cultured primary fibroblasts and early stage embryos. A simple SB transposon based co-selection strategy enabled enrichment for TALEN modified cells and efficient isolation of modified clones: single gene mono- and bi-allelic modification was induced in up to 54% and 17% of colonies respectively. It also enabled isolation of colonies harboring large chromosomal deletions (10% of colonies) and inversions (4%) after treatment with two TALEN pairs. We derived miniature swine models of familial hypercholesterolemia from LDLR mono- and bi-allelic TALEN-knockout fibroblasts. We next utilized TALEN and CRISPR/Cas9 stimulated homology-directed repair (HDR) to edit genes with oligonucleotide, plasmid, and rAAV templates without any drug selection. We first introgressed a bovine POLLED allele into horned dairy bull fibroblasts to circumvent manual dehorning. We also introduced single-nucleotide alterations or small indels into 14 additional genes in pig, cattle and sheep, into 10-50% of cells from fibroblast populations treated with TALEN mRNA and oligonucleotides. Up to 67% of propagated colonies harbored the intended edits and over one-half were homozygous. Some edits were naturally occurring SNP alleles, equivalent to non-meiotic inter- or intra-species introgression of valuable alleles. We created pig models for infertility and colon cancer from colonies with TALEN-HDR knockout alleles in DAZL and APC.
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    Production of induced regulatory T-cells through CRISPR/Cas9-based gene editing
    (2018-12) Tschann, Madison
    Regulatory T-cells (Tregs) are a subset of T-cells essential for maintaining immune tolerance and their dysregulation has been found to have a central role in the progression of various autoimmune diseases. The transplantations of Treg as a form of immune therapy has and continues to be an attractive method for the treatment of such disease based on their immuno-modulatory properties. Despite its potential, Treg adoptive cell transfer therapy is hampered by limited isolation efficiency due to low frequencies in human peripheral blood and poor in vitro expansion of a pure population. Herein, a novel CRISPR/Cas9 based technique is described utilizing AAV incorporation of strong transcriptional elements into the promoter region of the Treg master transcription factor, FOXP3, to upregulate expression in isolated primary T-cells and drive them toward a Treg phenotype.
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    Using Genetics to Understand and Overcome CART Cell Resistance and Toxicities
    (2022-04) Cox, Michelle
    Chimeric antigen receptor T (CART) cell therapy is an engineered cellular therapy that redirects T cells to cancer cells expressing certain antigens. CD19-directed CART cell therapy is the most advanced CART cell therapy in the clinic and is currently approved for the treatment of different B cell malignancies. However, the wider application of CART cell therapy in hematological malignancies is limited by its toxicities and lower rates of durable remission. Through translational and correlative science, large data analysis, bioinformatics, and advances in synthetic biology, we have learned that a predominant mechanism of CART cell therapy resistance is the immunosuppressive tumor microenvironment (TME). The main objective of this work was to understand how the TME impacts CART cell functions and to create inhibition-resistant CART cells utilizing genetic sequencing and synthetic biology tools. Specifically, we aimed to investigate the mechanisms by which 1) granulocyte-macrophage colony-stimulating factor (GM-CSF) and 2) leukemic extracellular vesicles (EVs) impact CART cell functions.In the first part of this work, we have discovered that GM-CSF directly impacts CART cells through modulation of their activation pathways. We have previously demonstrated that GM-CSF contributes to myeloid cell activation and to the development of toxicities after CART cell therapy. Using CRISPR/Cas9, we generated GM-CSFKO CART19 cells and demonstrated their reduced production of GM-CSF. GM-CSFKO CART19 cells demonstrated enhanced proliferation and superior anti-tumor activity in preclinical models, suggesting a direct effect of GM-CSF disruption on CART cells, independent of its known modulation of myeloid cells. To investigate the mechanism of this enhanced efficacy, we first ruled out off-target editing by performing whole exome sequencing. We then interrogated the transcriptome of GM-CSFKO CART19 cells, which showed a distinct gene expression profile suggesting alteration of activation pathways. We validated this immunophenotype on a protein level and its effect on CART cell activation and functions in vivo. In the second part of this work, we discovered a novel mechanism of resistance to CART cell therapy through their inhibition by tumor-derived extracellular vesicles. In this work, we used chronic lymphocytic leukemia (CLL) as a model to interrogate these interactions. The immunosuppressive microenvironment in CLL is well known to inhibit effector immune cells and in part may be related to the abundance of circulating EVs bearing immunomodulatory properties. We hypothesized that CLL-derived EVs contribute to CART cell dysfunction. To test this hypothesis, we first enumerated and immunophenotyped circulating EVs from platelet-free plasma in untreated patients with CLL. We determined their interaction with CART19 cells and found that CLL-derived EVs impair normal donor CART19 antigen-specific proliferation and killing. Our mechanistic studies demonstrated that CLL-derived EVs induce a state of T cell dysfunction characterized by functional, immunophenotypical, and transcriptional hallmarks of exhaustion and this dysfunction is more specific for PD-L1high EVs. In conclusion, we demonstrate that 1) CRISPR/Cas9 GM-CSF knockout in CART cells modulates their activation and enhances overall expansion and 2) leukemic EVs induce significant CART19 cell dysfunction by altering exhaustion pathways. GM-CSFKO CART19 is a novel CART cell therapy that is potentially less toxic and more effective than current CART19. The knowledge that leukemic EVs induce CART cell dysfunction paves the way for future studies of EV phenotype and cargo, which can ultimately lead to new strategies to predict outcomes and implement individualized CART cell therapy.